Dorsal root ganglia (DRG) cultures were produced from E16 to E17 Sprague–Dawley rats and maintained as described previously with minor modifications [37 (link)]. (See Online Resource 1: Supplemental Methods for additional details).
DRG cultures were incubated with either capsaicin (Sigma) or control solution (DMSO, Sigma) for 15 min, replenished with fresh medium, and further incubated for 1.5 or 48 h to evaluate axonal swellings and axonal loss, respectively. Serial capsaicin concentrations of 1, 10, 100 μM and 1 mM were tested to optimize treatment conditions and 100 μM capsaicin was used in subsequent experiments because it induced axonal ovoid formation and alterations in axonal mitochondrial behavior without causing axonal loss during the 15-min incubation period. In a set of experiments, 10 μM capsazepine (CZP, Sigma), a capsaicin antagonist, was added to DRG cultures for 10 min before the addition of capsaicin or DMSO.
DRG cultures were incubated with either capsaicin (Sigma) or control solution (DMSO, Sigma) for 15 min, replenished with fresh medium, and further incubated for 1.5 or 48 h to evaluate axonal swellings and axonal loss, respectively. Serial capsaicin concentrations of 1, 10, 100 μM and 1 mM were tested to optimize treatment conditions and 100 μM capsaicin was used in subsequent experiments because it induced axonal ovoid formation and alterations in axonal mitochondrial behavior without causing axonal loss during the 15-min incubation period. In a set of experiments, 10 μM capsazepine (CZP, Sigma), a capsaicin antagonist, was added to DRG cultures for 10 min before the addition of capsaicin or DMSO.